Characterization of the channel-pores formed by Bacillus thuringiensis Cry46Ab toxin in planar lipid bilayers

  • Akira Sakakibara
  • So Takebe
  • Toru Ide
  • Tohru HayakawaEmail author
Original Research Paper


Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab, therefore, is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 ± 2.7 pS in 150 mM NaCl and 24.2 ± 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K+ to Cl was approximately 4, and the preferences for cations were K+ > Na+, K+ > Ca2+, and Ca2+ > Na+. A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins.


Bacillus thuringiensis Cry46Ab toxin Planar lipid bilayer Single-channel analysis Calcein release assay 



C. pipiens eggs were kindly supplied by the Research and Development Laboratory at Dainihon Jochugiku Co., Ltd., Osaka, Japan. This work was supported in part by JSPS KAKENHI Grant number JP18K05675.


  1. Abrami L, Fivaz M, van der Goot FG (2000) Adventures of a pore-forming toxin at the target cell surface. Trends Microbiol 8:168–172. CrossRefGoogle Scholar
  2. Akiba T, Abe Y, Kitada S, Kusaka Y, Ito A, Ichimatsu T, Katayama H, Akao T, Higuchi K, Mizuki E, Ohba M, Kanai R, Harata K (2009) Crystal structure of the parasporin-2 Bacillus thuringiensis toxin that recognizes cancer cells. J Mol Biol 386:121–133. CrossRefGoogle Scholar
  3. Ballard J, Sokolov Y, Yuan WL, Kagan BL, Tweten RK (1993) Activation and mechanism of Clostridium septicum alpha toxin. Mol Microbiol 10:627–634. CrossRefGoogle Scholar
  4. Ben-Dov E (2014) Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins 6:1222–1243. CrossRefGoogle Scholar
  5. Benz R, Popoff MR (2018) Clostridium perfringens enterotoxin: the toxin forms highly cation-selective channels in lipid bilayers. Toxins (Basel) 10:E341. CrossRefGoogle Scholar
  6. Benz R, Janko K, Läuger P (1979) Ionic selectivity of pores formed by the matrix protein (Porin) of Escherichia coli. Biochem Biophys Acta 551:238–247. CrossRefGoogle Scholar
  7. Boonserm P, Davis P, Ellar DJ, Li J (2005) Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 348:363–382. CrossRefGoogle Scholar
  8. Boonserm P, Mo M, Angsuthanasombat C, Lescar J (2006) Structure of the functional from of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstorm resolution. J Bacteriol 188:3391–3401. CrossRefGoogle Scholar
  9. Chakraborty T, Schmid A, Notermans S, Benz R (1990) Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus. Infect Immun 58:2127–2132Google Scholar
  10. Cohen S, Albeck S, Ben-Dov E, Cahan R, Firer M, Zaritsky A, Dym O (2011) Cyt1Aa toxin: crystal structure reveals implications for its membrane-perforating function. J Mol Biol 413:804–814. CrossRefGoogle Scholar
  11. Crickmore N, Bone EJ, Williams JA, Ellar DJ (1995) Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131:249–254. Google Scholar
  12. de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199. CrossRefGoogle Scholar
  13. Fernández LE, Pérez C, Segovia L, Rodríguez MH, Gill SS, Bravo A, Soberón M (2005) Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II. FEBS Lett 579:3508–3514. CrossRefGoogle Scholar
  14. Georghiou GP, Wirth MC (1997) Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl Environ Microbiol 63:1095–1101Google Scholar
  15. Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz JL, Brousseau R, Cygler M (1995) Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation. J Mol Biol 254:447–464. CrossRefGoogle Scholar
  16. Hayakawa T, Kanagawa R, Kotani Y, Kimura M, Yamagiwa M, Yamane Y, Takebe S, Sakai H (2007) Parasporin-2Ab, a newly isolated cytotoxic crystal protein from Bacillus thuringiensis. Curr Microbiol 55:278–283. CrossRefGoogle Scholar
  17. Hayakawa T, Sakakibara A, Ueda S, Azuma Y, Ide T, Takebe S (2017) Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture. Insect Biochem Mol Biol 87:100–106. CrossRefGoogle Scholar
  18. Ito A, Sasaguri Y, Kitada S, Kusaka Y, Kuwano K, Masutomi K, Mizuki E, Akao T, Ohba M (2004) A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells. J Biol Chem 279:21282–21286. CrossRefGoogle Scholar
  19. Knapp O, Stiles B, Popoff MR (2010) The aerolysin-like toxin family of cytolytic, pore-forming toxins. Open Toxinol J 3:53–68. CrossRefGoogle Scholar
  20. Knowles BH, Ellar DJ (1987) Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity. Biochim Biophys Acta 924:509–518. CrossRefGoogle Scholar
  21. Likitvivatanavong S, Aimanova KG, Gill SS (2009) Loop residues of the receptor binding domain of Bacillus thuringiensis Cry11Ba toxin are important for mosquitocidal activity. FEBS Lett 583:2021–2030. CrossRefGoogle Scholar
  22. Melton JA, Parker MW, Rossjohn J, Buckley JT, Tweten RK (2004) The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin. J Biol Chem 279:14315–14322. CrossRefGoogle Scholar
  23. Pérez C, Fernandez LE, Sun J, Folch JL, Gill SS, Soberón M, Bravo A (2005) Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. Proc Natl Acad Sci USA 102:18303–18308. CrossRefGoogle Scholar
  24. Petit L, Maier E, Gibert M, Popoff MR, Benz R (2001) Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers. J Biol Chem 276:15736–15740. CrossRefGoogle Scholar
  25. Poncet S, Delécluse A, Klier A, Rapoport G (1995) Evaluation of synergistic interactions among the CryIVA, CryIVB, and CryIVD toxic components of B. thuringiensis subsp. israelensis crystals. J Invertebr Pathol 66:131–135. CrossRefGoogle Scholar
  26. Puntheeranurak T, Uawithya P, Potvin L, Angsuthanasombat C, Schwartz JL (2004) Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1–alpha5 fragment. Mol Membr Biol 21:67–74. CrossRefGoogle Scholar
  27. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813Google Scholar
  28. Schwartz JL, Garneau L, Savaria D, Masson L, Brousseau R, Rousseau E (1993) Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers. J Membr Biol 132:53–62. CrossRefGoogle Scholar
  29. Shai Y, Bach D, Yanovsky A (1990) Channel formation properties of synthetic pardaxin and analogues. J Biol Chem 265:20202–20209Google Scholar
  30. Sher D, Fishman Y, Zhang M, Lebendiker M, Gaathon A, Mancheño JM, Zlotkin E (2005) Hydralysins, a new category of β-pore-forming toxins in Cnidaria. J Biol Chem 280:22847–22855. CrossRefGoogle Scholar
  31. Slatin SL, Abrams CK, English L (1990) Delta-endotoxins form cation-selective channels in planar lipid bilayers. Biochem Biophys Res Commun 169:765–772. CrossRefGoogle Scholar
  32. Tabashnik BE, Cushing NL, Finson N, Johanson MW (1990) Field development of resistance of Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol 83:1671–1676. CrossRefGoogle Scholar
  33. Von Tersch MA, Slatin SL, Kulesza CA, English LH (1994) Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide. Appl Environ Microbiol 60:3711–3717Google Scholar
  34. Wirth MC, Georghiou GP, Federici BA (1997) CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc Natl Acad Sci USA 94:10536–10540. CrossRefGoogle Scholar
  35. Wirth MC, Park HW, Walton WE, Federici BA (2005) Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus. Appl Environ Microb 71:185–189. CrossRefGoogle Scholar
  36. Wirth MC, Walton WE, Federici BA (2012) Inheritance, stability, and dominance of cry resistance in Culex quinquefasciatus (Diptera: Culicidae) selected with the three cry toxins of Bacillus thuringiensis subsp. israelensis. J Med Entomol 49:886–894CrossRefGoogle Scholar
  37. Wu D, Johnson JJ, Federici BA (1994) Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis. Mol Microbiol 13:965–972. CrossRefGoogle Scholar
  38. Xu C, Wang BC, Yu Z, Sun M (2014) Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins. Toxins (Basel) 6:2732–2770. CrossRefGoogle Scholar

Copyright information

© The Japanese Society of Applied Entomology and Zoology 2019

Authors and Affiliations

  1. 1.Graduate School of Interdisciplinary Science and Engineering in Health SystemsOkayama UniversityOkayamaJapan
  2. 2.Graduate School of Biology-Oriented Science and TechnologyKindai UniversityKinokawaJapan

Personalised recommendations